DESCRIPTION provided by applicant The EPA maintains a Toxics Release Inventory of over toxic chemicals that are disposed or released from more than industrial sites in the United States The Department of Health and Human Services and USDA maintain a list of over select agents and toxins that pose a severe risk to both human animal and plant health These compounds represent only a fraction of the toxins and toxicants at environmental concentrations that pose substantial risk to human health At present assays that assess the impact of exposure to stem cell differentiation and programming are not capable of the throughput necessary to stay apace the speed of new toxicants entering the environment Such measurements are critical to our understanding of the risks these agents represent and to our ability to moderate or eliminate those risks The goal of this project is the development of instrumentation capable of detecting toxicant effects on stem cell differentiation processes in a sensitive high content assay based on the detection of changes in the differentiation profile of stem cells exposed to these toxicants Since stem cells are critical elements of embryonic development and for the ongoing maintenance of adult tissues an effective assessment of their function will be important of our understanding of environmental risks for autoimmune disease immunodeficiencies and neoplasia to name only a few of the health risks when stem cell behavior is altered The assay is based on instrumentation that can measure grating coupled surface plasmon resonance GCSPR and grating coupled surface plasmon coupled emissions SPCE This assay system will incorporate a reliable fluidic system and a simple to use sensor chip array that can be used for assessment of toxicant effects on stem cell differentiation and may ultimately also have value as a point of care diagnostic
PUBLIC HEALTH RELEVANCE There is a critical need for highly sensitive and specific techniques capable of defining the risk levels to human health that result from exposure to a broad range of toxicants found in the environment To address this need we propose to build upon our extensive experience with microarray Surface Plasmon Resonance SPR systems to develop a high content instrument platform that can assess the impact of toxicant exposure on stem cell differentiation and is not constrained by the important limitations of existing technologies This multi mode multi fluor SPR analysis will identify and quantitate the effects o toxicant exposure on stem cell functioning without foreknowledge of the contaminantandapos s identity or toxic concentration profile The technology will require small sample volumes and will be suitable for use in both research and clinical laboratories and with further development will be extensible to a point of care environment

An instrument for measuring and analyzing surface plasmon resonance (SPR) and/or surface plasmon coupled emission on an electro-optic grating-coupled sensor surface is described herein. The sensor chip achieves SPR through a grating-coupled approach, with variations in the local dielectric constant at regions of interest (ROI) at the sensor surface detected as a function of the intensity of light reflecting from these ROI. Unlike other grating-based approaches, the metal surface is sufficiently thin that resonant conditions are sensitive to dielectric constant changes both above and below the metal surface (like the Kretschmann configuration). Dielectric constant shifts that occur as mass accumulates on the surface can be returned to reference intensities by applying voltage across the underlying electro-optic polymer. Approaches to the development of the sensor surfaces are described, as are software and hardware features facilitating sample handling, data gathering, and data analysis by this solid-state approach.

DESCRIPTION provided by applicant Aberrant neuronal connectivity is associated with a number of neuropathological and neuropsychiatric diseases disorders Inefficient developmental connectivity misdirected connectivity or disrupted and degenerated connectivity can account for losses of normal neuronal communication and subsequent losses of mental or physical health Altered expression of axonal guidance proteins neuroinflammation neuronal toxicity and glial disturbances may be involved in inadequate neuronal connectivity The goal of the work described in this application is to develop an instrument platform that can both enumerate development or loss of axons and the kinetics of these changes in response to molecular and cellular modifiers The proposed grating coupled surface plasmon resonance GCSPR and grating coupled surface plasmon coupled emission GCSPCE instrumentation with a microflow biosensor chip will be developed and validated for quantification of developmental axonal outgrowths among punches of brain regional sections as well as loss of connections previously established Up to five mm punches can be placed on the cm biosensor gold chip precoated with extracellular matrix proteins and specific molecules guidance proteins or cells microglia spotted at specific locations Lack or loss of axonal connections will be kinetically monitored by SPR and presynaptic and postsynaptic antigens can be assayed by SPCE with fluorochrome conjugated antibodies to neuronal antigens The geometrical placement of specific brain regions will be identified so that preferential regional interactions can be quantified The planned device to assess neuronal circuitry is built upon extensive experience with SPR microarrays that can characterize presence of single cells and their released products The work described will produce instrumentation capable of providing a more complete and coherent picture of differential neuronal connectivities to provide a more comprehensive view of neuronal communications which will reveal new opportunities for therapeutic interventions
PUBLIC HEALTH RELEVANCE The proposed surface plasmon resonance SPR assessment of axonal outgrowth between brain regions on a biosensor chip takes a step beyond what is currently possible with microscopic techniques With the instrumentation and software the differential kinetics of axonal projections toward certain brain regions is measured by SPR and coupled fluorescence Additionally proteins and cells spotted at precise areas on the chip can be evaluated for their influences and modifiers can be added or removed from the flowcell with microfluidic exchanges

DESCRIPTION (provided by applicant): The proposed work will result in a characterized prototype instrument that will facilitate the identification of a broad range of analytes in small volumes of human blood, urine, saliva and other readily obtainable biological fluids with little sample preparation. The proposed instrument will incorporate two related analytical approaches, grating-coupled surface Plasmon resonance analysis and surface plasmon-enhanced fluorescence to provide a broad dynamic range coupledwith highly sensitive measurements. Analyte selectivity is achieved through the use of capture ligands e.g. antibodies, aptamers, etc. Sample volumes required for the proposed prototype are on the order of 0.3 to 0.5 ml. The proposed instrument will be designed to perform multiple (approx.1000) analyses simultaneously. Gold-clad superparamagnetic nanobeads will be used to increase the selectivity of measurements by transporting analyte to capture sites in a controlled magnetic field. The instrument will becharacterized through the use of two commonly encountered toxicants and a bacterial toxin. Software-controlled automation will make the instrument easy to use and suitable for use out of the laboratory. The instrument will provide an inexpensive and efficient way to directly identify or monitor suspected toxicants. PUBLIC HEALTH RELEVANCE: There is a critical need for highly sensitive and specific techniques capable of defining the exposure levels of human and animal populations to the broad rangeof toxins and toxicants found in the environment. We propose to develop an instrument that will meet this need without many of the limitations that constrain existing technologies. The proposed approach will perform large number (up to a thousand) simultaneous analyses in less than hour. The analyses will identify and quantitate the presence or absence of hundreds of potential toxicants that a patient might encounter without foreknowledge of the contaminant's identity. The technology upon which the proposedinstrumentation is based is suitable for use in a clinical laboratory and, with further development extendable to a point-of-care instrument. The instrument will require small sample volumes of biological fluids such as blood, urine or saliva.

DESCRIPTION (provided by applicant): The objective of this Phase I SBIR proposal is the development of a novel instrumentation platform based on Surface Plasmon Resonance (SPR) that if validated would greatly increase the utility of this label-free technology to researchers utilizing high-content assays in systems biology investigations. The benefits of this platform derive from a change in detection paradigm that permits solid-state measurements of SPR shifts as a function of voltage. By moving away from an angle-scanning or wavelength-scanning approach, the proposed proprietary electro-optic grating-coupled SPR platform (EOSPR) would investigate changes in bound mass at as many as 100,000 regions of interest simultaneously. The sensitivity increase over current instrumentation is expected to improve over current label-free state-of-the-art in that data collection will be significantly more rapid in the proposed systm than in other high-content devices. The inclusion of additional data into the averaged measurement implies a greater signal-to-noise ratio, but further advantages are available to the EOSPR system that would not be viable in other SPR platforms. A signal-chopping scheme that could only be implemented on a system that scans as rapidly as the EOSPR device is expected to reduce the noise of the instrumentation. The combination of better signal and reduced noise is expected to lead to increases in label- free detection sensitivity that could approach that of label-based techniques. In addition, sincescans of the SPR curves could be completed much more rapidly than in classic devices, the direct measure of more rapid binding events becomes possible. The steps that will be required to verify if this technology can be turned into a commercial product include: modeling of the proposed chip, fabrication the chip, modifying an existing SPR instrument that was previously designed by Ciencia, and conducting proof-of-concept experiments on the prototype system. The objectives to meet these tasks are outlined within the body of this proposal, and strategic consulting arrangements and subcontracts will be employed to maximize the possibility of success for this potentially high-impact project. PUBLIC HEALTH RELEVANCE: Current trends in biomedical research imply that the future of medicine will rely upon ever-increasing degrees of personalized therapy. As the number of understood disease states and therapeutic options increase, so does the amount of diagnostic information necessary to make an informed decision. The proposed EOSPR technology aims to aid systems biology researchers in elucidating and understanding these complex molecular mechanisms by filling the need for a sensitive, label-free, and high-content assay that is affordable enough for widespreadadoption.

DESCRIPTION (provided by applicant): Polymicrobial infections are of great concern to the research community because they have a dramatic impact on the productivity research groups, on the reliability of experimental results, and on the progress that can be made in unraveling fundamental biological questions that underpin our understanding of human health issues. The diagnosis of these infections is currently done using a combination of techniques (e.g. ELISA, PCR, Flow cytometry) that are laborious, costly, and time consuming. As a consequence, routine colony surveillance represents a significant impediment to research. We propose to develop a high content assay based on the physical phenomenon of surface plasmon resonance (SPR) done in two modalities (grating coupled surface plasmon resonance; GCSPR and grating coupled surface plasmon coupled emission; GCSPCE). The sensor chip employed in this system will use two orthogonal holographic diffraction gratings to enable simultaneous sensor chip interrogation inboth modes. As a consequence, we anticipate reaching high levels of sensitivity (lt 30 CFU/ml) and a large dynamic range (5 to 6 logs). Because the system is capable of measuring more than a thousand different analytes from a biological sample using simple fluidics and an inexpensive chip architecture, this system will be capable of near real-time measurements of biological samples with a low labor demand and at dramatic savings. Development of this diagnostics system will have future relevance in similardiagnoses in agricultural animals and in human populations. The goal is to create a T cell analysis system that offers high content screening, multi-parameter characterization capability and incorporates state-of- the-art integrated sample handling for ease-of-use. At present, there are no systems available that offer the breadth of capabilities, the simplicity of use and limited sample size requirements as the proposed analysis system. PUBLIC HEALTH RELEVANCE: Polymicrobial infections in researchmay affect the colony health and productivity, and may compromise the accuracy of experimental results. As a consequence, research that is designed to enlarge our understanding of human disease and to develop better therapies and preventative treatments isat risk. This application aims to develop a high content assay system that is at once able to provide rapid, accurate, and sensitive measurements of infectious agents in an inexpensive and easy to use format. Use of this system will protect the integrityof our research infrastructure, and may ultimately be adaptable to diagnostic needs in agricultural herds and in the human population.

An instrument for measuring and analyzing surface plasmon resonance on a sensor surface has a polarized light source optically connected to the sensor surface by a plurality of optical elements, including in one embodiment an optical telescope that transfers light from a rotatable reflecting surface to the sensor surface. Selective positioning of a cylindrical lens into a first position within the path of light transforms collimated light to a rectangular wedge that is incident upon the sensor surface at numerous angles. In another embodiment, the light source is operated as a laser to excite fluorescence on the sensor surface and the fluorescence is selectively directed to a detector by appropriate optical elements positioned in specific configurations.

An instrument for measuring and analyzing surface plasmon resonance (SPR) and/or surface plasmon coupled emission on an electro-optic grating-coupled sensor surface is described herein. The sensor chip achieves SPR through a grating-coupled approach, with variations in the local dielectric constant at regions of interest (ROI) at the sensor surface detected as a function of the intensity of light reflecting from these ROI. Unlike other grating-based approaches, the metal surface is sufficiently thin that resonant conditions are sensitive to dielectric constant changes both above and below the metal surface (like the Kretschmann configuration). Dielectric constant shifts that occur as mass accumulates on the surface can be returned to reference intensities by applying voltage across the underlying electro-optic polymer. Approaches to the development of the sensor surfaces are described, as are software and hardware features facilitating sample handling, data gathering, and data analysis by this solid-state approach.

DESCRIPTION (provided by applicant): Type 1 Diabetes (T1D) affects more than 1 million people in the United States Source ADA http://www.diabetes.org/about-diabetes.jsp and is usually diagnosed in children and can lead to blindness, heart disease and kidney failure. A major focus of intervention for T1D is on the detection and characterization of auto-reactive T cells, which play a central role in the attack on insulin producing islet cells. For medical intervention for T1D to be most effective, the disease should be detected and treated before the onset of symptoms. Current methods to identify the targets of auto-reactive T1D-specific T cells are slow, technically demanding, labor- and reagent-intensive, and consume large numbers of T cells to test limited numbers of targets. Sample size is an extremely important consideration in T1D and is often a limiting factor when testing clinical samples, especially as young children and adolescents are unable to give large samples. The objective of this SBIR application is to develop an automated T cell analysis assay to identify and functionally characterize auto-reactive T1D antigen-specific CD4+ and CD8+ T cells capable of testing hundreds to thousands of targets simultaneously using as little as 2ml of patient sample. The proposed system is based upon integrating patent pending MHC-peptide array technology with an automated flow cell detection and analysis system. The innovative approach to this project will combine a self-contained sample cartridge in which antigen-specific CD4+ and CD8+ auto-reactive T cells bind to high avidity MHC-peptide monomers and are detected via surface plasmon resonance with correlated highly sensitive surface plasmon resonance-enhanced detection of cytokines secreted by identified cellsubpopulations. The goal is to create a T cell analysis system that offers high content screening, multi-parameter characterization capability and incorporates state-of- the-art integrated sample handling for ease-of-use. At present, there are no systems available that offer the breadth of capabilities, the simplicity of use and limited sample size requirements as the proposed T cell analysis system using MHC-peptide arrays in the fight against T1D. PUBLIC HEALTH RELEVANCE: Type 1 Diabetes (T1D) isan autoimmune disease usually diagnosed in children where T cells, part of the body's own immune system, attack pancreas cells that make insulin. Detecting auto-reactive T cells with current methods is impractical, time consuming and requires large patient samples. This application is to develop a high throughput T cell analysis system to detect and characterize auto-reactive T1D T cells using substantially smaller samples. Knowledge gained by the use of this T cell analysis system may lead to the faster discovery of T1D T cell targets, the ability to better monitor T1D therapies in clinical trils and the potential for earlier diagnosis of T1D.

DESCRIPTION (provided by applicant): The long-term objective of the proposed effort is to develop an autonomous clinical diagnostics system for monitoring normal and diseased immune function that occurs in the context of rheumatoid arthritis, infection, inflammation, asthma, autoimmune disease, and neoplasia. This system will be a microarray-based bioassay platform that specifically captures distinct cell types and soluble macromolecules from a patient's blood sample onto spatially separate regions on a biosensor chip. Initial capture of macromolecules and cells can be quantified to assess the presence of specific subpopulations of leukocytes, cytokines, antibodies and other immunologically relevant molecules. Subsequent culture of the captured cells under different conditions can be used to measure the production of specific cell products in real time to assess immune cell functional capacities. The technology allows for highly parallel measurements that result in the capability to perform hundreds of individual functional cellular assays simultaneously without the use of molecular labels. The system will enable assessment of patients' immune capacity in near-real time using microliter-scaled samples of blood readily harvested with minimally invasive procedures (e.g., fingerstick capillary blood sampling). Such real-time measurements will be crucial for the ongoing monitoring of disease progression as well as the evaluation of prophylactic treatments and therapeutic manipulations that sustain and support immune mechanisms. PUBLIC HEALTH RELEVANCE: This project will develop tools that will enable improved diagnostics for rheumatoid arthritis, autoimmune diseases, and for infectious diseases.